Method for producing cephalexin

ABSTRACT

The invention relates to a process for the preparation of cephalexin with the aid of a penicillin amidase immobilized on a crosslinked hydrophilic copolymer which has binding activity for ligands having nucleophilic groups and is in bead form.

The invention relates to a process for the preparation of cephalexinwith the aid of a penicillin amidase immobilized on a crosslinkedhydrophilic copolymer which has binding activity for ligands havingnucleophilic groups and is in bead form.

PRIOR ART

Processes for the synthesis of semisynthetic beta-lactam antibiotics byacylation of a beta-lactam residue (beta-lactam nucleus) with anactivated side chain, such as, for example, an amide or an ester, usingenzyme penicillin acylase (penicillin amidase) are well known to theskilled worker.

In most of these cases, the enzyme if bound to a solid, water-insolublecarrier and is subsequently brought into contact in aqueous solutionwith the beta-lactam nucleus and the activated side chain.

The disadvantage of the processes disclosed to date is the fact that theratio of the synthesis of the desired compound by the enzyme comparedwith the hydrolysis of the activated side chain to worthless side chainacids and also to the hydrolysis of the desired product, the so-calledS/H value, is often unfavorable and makes economic production difficult.

WO 93/12250 discloses that in the synthesis of the semisyntheticbeta-lactam antibiotics cephadroxil and cephalexin by E. coli penicillinamidase immobilized on Eupergit® (Röhm GmbH & Co. KG, Darmstadt,Germany, see also comparative example 1) the SOH value can bebeneficially influenced by the choice of the reaction conditions. Aninfluence of the nature of the carrier material is, on the other hand,not disclosed. The particular disadvantage of the process disclosed inWO 93/12250 is that the cephalexin is isolated from the reaction mixturein a complex with beta-naphthol, so that subsequent purification stepsare necessary, and product losses occur.

Attempts have therefore been made to develop optimized carriermaterials. Thus, WO 97/04086 discloses an E. coli penicillin amidasewhich is immobilized on a carrier material composed of a swelling agentand a polymer having free amino groups, and the use thereof forpreparing beta-lactam derivatives. The disclosed process for preparingcephalexin has, however, the disadvantage that the beta-lactam nucleus7-aminodeacetoxycephalosporanic acid (7-ADCA) is employed in athree-fold molar excess compared with the activated side chainD-phenylglycinamide (PGA). If the above-stoichiometry amounts ofbeta-lactam nucleus are employed, the nucleus must be recycled on theindustrial scale in order to be able to operate economically. This iscostly and leads to losses of yield. In addition, this circumstance alsoleads to impurities in the product because the nucleus is unstable.

EP 0 730 035 likewise discloses the preparation of cephalexin on aspecific carrier in acceptable yields. However, the particle size of thecarrier material used (Emphaze™) is only 60-80 μm. This is a greatdisadvantage for industrial applications. Thus, chromatography columnspacked with such material have only a low flow rate.

A carrier material for enzymes is described in DE 198 04 518. It ismentioned that the material can be used for enzymatic synthesis ofamoxycillin and ampicillin. Suitability for synthesizing cephalexinusing immobilized penicillin amidase is not mentioned.

Problem and Solution

In view of the prior art discussed above, the present invention wastherefore based on the problem of providing an improved process for thesynthesis of cephalexin which overcomes the above-mentioneddisadvantages.

This process was particularly intended to make it possible to achieve afavorable S/H value. It was further intended that the particle size ofthe carrier material used be from 120 to 250 μm, which is favorable forindustrial processes.

This problem is solved by the process defined in claim 1. Preferredembodiments of the process of the invention are defined in the dependentclaims which refer back to claim 1.

In particular, it is possible in a technically simple manner which couldnot have been predicted to solve the above problem by crosslinkedhydrophilic carrier polymer materials which have binding activity forligands having nucleophilic groups, are in bead form and can be preparedby inverse bead polymerization of a monomer phase which consist ofmonomers and a diluent, where the monomers present are

-   -   (a) 5-40% by weight of hydrophilic monomers which are capable of        free-radical polymerization, have a vinyl group and form at        least 10% strength aqueous solutions at room temperature,    -   (b) 30-50% by weight of monomers which are capable of        free-radical polymerization and have a vinyl group and an        additional functional group which is able to enter into covalent        bonds in a polymer-analogous reaction with the nucleophilic        groups of the ligands,    -   (c) 20-60% by weight of crosslinking monomers which are capable        of free-radical polymerization and have two or more        ethylenically unsaturated polymerizable groups,        with the proviso that a), b) and c) add up to 100% by weight,        and the diluent used is a mixture of methanol and water in the        ratio from 1:1.0 to 1:4.0, where the monomer phase is dispersed        to droplets in a continuous phase composed of an organic solvent        composed of an aliphatic hydrocarbon having 5-7 carbon atoms,        where the ratio of monomer phase to continuous phase is from        1:2.0 to 1:4.0, and in this form undergo free-radical        polymerization in the presence of a polymerization initiator and        of a protective colloid, with the proviso that the ratio of the        monomers to the diluent is from 1:1.7 to 1:2.4, being coated        with penicillin amidase, and these coated carriers being brought        into contact with an aqueous solution which comprises    -   (i) 7-aminodeacetoxycephalosporanic acid and    -   (ii) D-phenylglycinamide.        in ratios of from 1:2 to 2:1, preferably 1.5:1 to 1:1.5,        particularly preferably in approximately equal molar ratios,        i.e. in ratios of from 1.2:1 to 1:1.2.

The carrier material used and the process for its preparation aredescribed in DE 198 04 518.

Implementation of the Invention

Preparation of the Carrier Material

Monomers

In order to ensure the hydrophilicity of the monomer mixture, it mustconsist predominantly of hydrophilic monomers. By hydrophilic monomersare meant those monomers which form at least 10% strength aqueoussolutions at room temperature and preferably comprise no ionic group orgroups which can be ionized by addition of acid or base.

Monomers a) are 5-40, 8-35, in particular 9-12, % by weight ofhydrophilic monomers which are capable of free-radical polymerizationand have a vinyl group which form at least 10% strength aqueoussolutions at room temperature.

Particularly suitable monomers a) are acrylamide and/or methacrylamide,with preference for methacrylamide. Further examples are hydroxyalkylesters of unsaturated polymerizable carboxylic acids, such ashydroxyethyl acrylate and hydroxyethyl methacrylate orN-vinylpyrrolidone.

Monomers b) are 30-50, preferably 35-45, % by weight of monomers whichare capable of free-radical polymerization and have a vinyl group and anadditional functional group, preferably an oxirane group (epoxy group),which is able to enter into covalent bonds in a polymer-analogousreaction with the nucleophilic groups of the ligands. Oxirane groups inparticular are suitable for binding ligands with retention of theirbiological activity.

Preferred monomers b) are glycidyl methacrylate and/or allyl glycidylether. It is particularly preferred for both monomers to be employedsimultaneously in approximately equal amounts.

Monomers c) are 20-60, in particular 25-55, particularly preferably40-55, % by weight of hydrophilic, crosslinking monomers which arecapable of free-radical polymerization and have two or moreethylenically unsaturated polymerizable groups. Preferred monomers c)are N,N′-methylenebisacrylamide or N,N′-methylenebismethacrylamide.N,N′-methylenebismethacrylamide is particularly preferred. It is alsopossible where appropriate to employ 0-10% by weight of furthercrosslinking monomers which are capable of free-radical polymerizationand have two or more ethylenically unsaturated polymerizable groups.Hydrophilic di(meth)acrylates are suitable, such as, for example,polyethylene oxide di(meth)acrylates.

Monomers a), b) and c) in each case add up to 100% by weight.

Diluents

The monomer phase consists of monomers a) to c) dissolved in a diluentwhich must be a mixture of methanol and water in the ratio from 1:1.0 to1:4.0. Particularly favorable mixing ratios for methanol and water arefrom 1:1.2 to 1:2.5, in particular from 1:1.3 to 1:1.7.

Ratio of Monomers to Diluent

The ratio of monomers to diluent is particularly critical. This must bein the range from 1:1.7 to 1:2.4, particularly preferably in the rangefrom 1.9 to 2.1.

Continuous Phase

A suitable continuous phase is an organic solvent which is an aliphatichydrocarbon having 4 to 7 C atoms. n-heptane is preferred, andcyclohexane is particularly preferred.

Monomer Phase/Continuous Phase Ratio

The ratio of the monomer phase to a continuous phase formed by theorganic solvent must be from 1:2.0 to 1:4.0, preferably between 1:2.8 to1:3.3.

Further Process Conditions

As further constituents, the suspended monomer phase comprises in amanner known per se polymerization initiators, with preference forsulfur-free initiators, and with particular preference for4,4′-azobis(4-valeric acid), and protective colloids (emulsifiers) suchas, for example a copolymer with 95 parts of n-butyl methacrylate and 5parts of 2-trimethylammoniumethyl methacrylate chloride having weightaverage molecular weights in the range from 30 000 to 80 000.

The bead polymerization (also referred to as suspension polymerization)is otherwise carried out in a known manner by, for example, introducingthe continuous phase and the protective colloid, and dispersing themonomer phase, in which the initiator is also present, with stirring,e.g. at 40 to 60° C., in the organic phase, and subsequently heating to60-70° C. The water/methanol mixture can be removed azeotropicallyalmost completely for example over a period of 6 hours. The mixture isallowed to react to the end for about 3-5 hours and is subsequentlycooled to room temperature. The resulting beads are filtered off withsuction and dried for example in vacuo for 12 hours. An alternativepossibility is also for the bead polymers to be filtered off and washedwith water. Drying is preferably carried out in a fluidized bed dryer,because solvent residues can be removed particularly efficiently in thisway. The resulting polymer beads (=carrier polymer material) have a sizein the range from 50 to 500 μm, in particular from 120 to 250 μm. By thebinding capacity is meant the enzymatic activity which can be reached onmaximum loading of the carrier polymer material with a particularenzyme. The binding capacity is expressed as penicillin amidase activityin units per g of carrier polymer beads [U/g moist]. The bindingcapacity of the carrier polymer beads of the invention with this methodof measurement is at least 220 [U/g moist].

The swellability of the polymer beads in water is expressed by theswelling index [ml moist/ml dry]. The carrier polymer beads of theinvention have a swelling index not exceeding 1.5.

Coating of the Carrier Material which can be Used According to theInvention

In the examples, the carrier material is coated at pH 7.5 in potassiumphosphate buffer. However, the skilled worker is aware that there is avery large number of other processes ensuring satisfactory coating.

Synthesis of Cephalexin

The precursors (i) 7-aminodeacetoxycephalosporanic acid and (ii)D-phenylglycinamide are employed in concentrations of 10-500 mM,preferably 50-300 mM and particularly preferably of 150-250 mM.

Advantageous Effects of the Invention

The process of the invention makes it possible to synthesize cephalexinwith a favorable S/H ratio (synthesis/hydrolysis). It is advantageous inthis connection that this is achieved by using a carrier material withparticle sizes in the range of 50-500 μm, in particular from 120 to 250μm. Better technical application properties are achieved in this way,e.g. higher flow rates in fixed bed reactors. The higher flow ratesresult in better space-time yields. The larger carrier particles alsohave advantages in batch processes, because they can be filtered offfaster. This in turn increases the space-time yield and thus thecommercial viability of the process.

EXAMPLES

(The following determination method is familiar per se to the personskilled in the area of porous carrier polymer materials and is detailedonly for the sake of completeness)

Determination of the binding capacity for penicillin amidase(=penicillin G acylase) from E. coli (EC 3.5.1.11).

a) Covalent Bonding of Penicillin Amidase to the Carrier PolymerMaterial

1 g of carrier polymer material are added to 1530 units of penicillinamidase in 5 ml of sterile 1 M potassium phosphate buffer pH 7.5 andincubated at 23° C. for 48 h.

The polymer beads are then put on a sintered-glass frit (porosity 2 or3) and washed twice with deionized water and then twice with 0.1 Mpotassium phosphate buffer pH 7.5, containing 0.05% ethyl4-hydroxybenzoate, by suction on the frit. The moist weight of theresulting beads loaded with penicillin acylase is determined.

b) Determination of the Binding Capacity

250-300 mg of moist carrier polymer material (polymer beads) coupled topenicillin amidase are put into 20 ml of a 2% strength penicillin Gsolution in 0.05 M potassium phosphate buffer pH 7.5, containing 0.05%ethyl 4-hydroxybenzoate, at 37° C. Liberated phenylacetic acid istitrated with 0.5 M NaOH while stirring continuously at a constant pH of7.8 for a period of 10 minutes, recording the NaOH used.

The polymer beads are then obtained as in a) by sucking 20 ml of 0.05 Mpotassium phosphate buffer pH 7.5, containing 0.05% ethyl4-hydroxybenzoate, through a glass frit, and the measurement is repeatedtwice.

c) Calculation of the Binding Capacity

The linear region of the measured curves (normally the region of 1-5min) is used as basis for the calculation and extrapolated to a 10 mininterval. The binding capacity is reported as penicillin amidase unitsper g of moist carrier polymer material (U/g moist). One unitcorresponds to one μmol of hydrolyzed penicillin G per minute(μmol/min); 1 l of 0.5 M NaoH is in this case equivalent to 500 μmol ofhydrolyzed penicillin G. (The water content of the carrier polymermaterial is approximately constant and can therefore be neglected.)

Comparative Example 1

1530 units of penicillin amidase from E. coli are dissolved in 6 ml ofsterile 1 M potassium phosphate buffer, pH 7.5. The solution is added to1 g of Eupergit® C (Röhm GmbH & Co. KG, Darmstadt, Germany), and theresulting suspension is incubated at 23° C. for 72 hours. The polymerbeads are collected on a sintered glass filter and washed with 0.1 Mpotassium phosphate buffer. The following cephalexin synthesis anddetermination of the S/H value follow examples 5 and 6, respectively.

Eupergit® C (a copolymer of N,N′-methylenebismethacrylamide, allylglycidyl ether and methacrylamide) and processes for its preparation aredescribed in DE-C 27 22 751, U.S. Pat. No. 490,713 and U.S. Pat. No.4,511,694.

Comparative Example 2

Following WO 97/04086, a cephalexin synthesis is carried out asdescribed in example 6 using the type A enzyme or type B enzymedisclosed therein.

Comparative Example 3

E. coli penicillin amidase is immobilized on Sepabeads® FP-EP orSepabeads® FP-EP/G (Resindion S.R.I., Milan, Italy) as shown incomparative example 1. The following cephalexin synthesis and thedetermination of the S/H value follow examples 5 and 6, respectively.

Sepabeads® FR-EP or Sepabeads® FP-EP/G is a highly polymeric crosslinkedacrylic copolymer having oxirane groups, like Eupergit®. The averageparticle size according to the manufacturer's information is 150-300 μm.

Comparative Example 4

Measurement of the flow rate of a chromatography column packed withEmphaze™ and the material which can be used according to the invention.

-   Material: Emphaze Ultralink™ Biosupport Medium, Lot#DC53515, Pierce,    average particle size according to the manufacturer's information    50-80 μm;    -   material which can be used according to the invention, average        particle size 208 μm.

Borosilicate glass chromatography columns with bottom glass frits andpolypropylene end caps (dimension of the column 1×20 cm) were testedempty for a comparable outflow rate. The carrier materials weresuspended in water overnight and then rinsed with water into theparticular column, and a packed bed with a height of 6.5 cm was obtainedby sedimentation and slow outflow of the liquid. Any voids were removedby gentle tapping. The columns were open at the top and had a constantwater column of 23 cm through inflow of water.

The flow rate was achieved only by the hydrostatic pressure. The flowrate was determined using a stopwatch and 10 ml graduated cylinder. Theflow rate determined for a preparation which can be used according tothe invention was 4.25 ml per minute. The flow rate determined forEmphaze™ was 0.71 ml per minute. The suitability of spherical particlesfor operating the fixed bed reactors improves as the flow rate increases(higher space-time yield). The pressure drop in fixed bed reactors canalso be calculated mathematically: K. Buchholz and B. Gödelmann in“Characterization of immobilized biocatalysts”, Dechema Monographs,volume 84, editor K. Buchholz, VCH Weinheim 1979, pages 128-129).

The better technical application properties of the material which can beemployed according to the invention for use in fixed bed reactors,compared with Emphaze™, is clearly evident.

Examples 1-3

Consistent Test Conditions in Examples 1-3:

An organic solvent, 3 g of a copolymer of 95 parts of n-butylmethacrylate and 5 parts of 2-trimethyl-ammoniumethyl methacrylatechloride as protective colloid and 5 g of dry ice are introduced into a2 l stirred flask with thermometer, water trap, reflux condenser,nitrogen-introduction tube. While stirring and passing nitrogen through,a monomer phase consisting of water and methanol in the ratio 1:1.5(example 1) or of formamide (examples 2 and 3) as diluent, and

-   -   10 g of methacrylamide,    -   20 g of allyl glycidyl ether,    -   20 g of glycidyl methacrylate and    -   50 g of methylenebismethacrylamide    -   and    -   2 g of 4,4′-azobis-4-cyanovaleric acid (as polymerization        initiator).        is dispersed in the organic phase at 50° C., and then heated to        boiling at 65-70° C. The mixture is incubated for about 6 h and        then cooled to room temperature. The resulting polymer beads are        filtered off with suction, washed and dried in a fluidized bed        dryer. The binding capacity for penicillin amidase [U/g moist]        and the swelling index is determined [ml moist/ml dry]        determined.

The essential test parameters and the results of examples 1-3 are to befound in the following table.

Example 1 Example 2 Example 3 (according to (comparative (comparativethe invention example) example) Organic solvent 952 g of 669 g of 530 gof (continuous cyclohexane cyclohexane n-heptane + 530 g phase) ofperchloro- ethylene Monomers in total 100 g 100 g 100 g Diluent 80 g of263 g of 264 g of methanol + 120 g formamide formamide of water (=1:1.5)Monomers + diluent 300 g 363 g 364 g (monomer phase) Monomer/diluent 1:21:2.63 1:2.64 ratio Monomer phase/ 1:3.2 1:1.8 1:2.9 continuous phaseratio Binding capacity 252 194 192 for penicillin amidase (1530 U) [U/gmoist] Swelling index  1.3  4.0  3.9 [ml moist/ml dry]

Example 4

Synthesis of Cephalexin

The reaction is carried out at 25° C. and pH 7.5 in a fixed bed reactor.10 ml of an aqueous solution of a mixture 0.2 M 7-ADCA and 0.2 MD-phenylglycinamide are passed through a column with 0.5 ml of theimmobilized penicillin amidase. During the reaction, the pH is keptconstant by adding HCl in an attached stirred reservoir container.Samples are taken and analyzed by HPLC as shown in example 5 atparticular time intervals.

Example 5

The products of the reaction from example 4 and of comparative examples1 to 3 were analyzed by HPLC using an RP-8 column (Merck KGaA,Darmstadt, Germany). The mobile phase used was sterile 67 mM potassiumphosphate buffer pH 7.5. Cephalexin was eluted with a 30% strength(volume/volume) aqueous methanol solution.

The cephalexin synthesis rate (V_(Ceph)) and the D-phenylglycinehydrolysis rate (V_(D-PhG)) was determined from the HPLC analysis, andthe S/H value (synthesis rate/hydrolysis rate (V_(Ceph)/V_(D-PhG)) ratiowas calculated therefrom. The results are shown in the following table:

Enzyme source Carrier/immobilizates V_(Ceph)/V_(D-PhG) E. coli accordingto the invention 4.6 E. coli Type A from WO 97/04086 3.3 (seecomparative example 2) E. coli Type B from WO 97/04086 3.3 (seecomparative example 2) E. coli Comparative example 1 3.5 E. coliComparative example 3 3.2 (Sepabeads FP-EP) E. coli Comparative example3 2.9 (Sepabeads FP-EP/G)

The value for the process of the invention is composed of 7 test seriescarried out in parallel, which resulted in the following S/H values:5.3, 4.7, 3.6, 4.6, 4.3, 5.2, 4.7;

The significant improvement of about 30% in the S/H value which can beachieved with the process of the invention compared with the comparativeexamples is clearly evident.

1. A process for the preparation of cephalexin, comprising: contactingan aqueous solution, comprising: (i) 7-aminodeacetoxycephalosporanicacid, and (ii) D-phenylglycinamide, in a ratio from 1:2 to 2:1, with acoated carrier, wherein the carrier comprises crosslinked hydrophiliccarrier polymer materials which are able to form covalent bonds in apolymer-analogous reaction with nucleophilic groups of ligands, are inbead form and can be prepared by inverse bead polymerization of amonomer phase which consists of monomers and a diluent, wherein themonomers are: (a) 5-40% by weight of hydrophilic monomers which arecapable of free-radical polymerization, have a vinyl group and form atleast 10% strength aqueous solutions at room temperature, (b) 30-50% byweight of monomers which are capable of free-radical polymerization andhave a vinyl group and an additional functional group which is able toenter into covalent bonds in a polymer-analogous reaction with thenucleophilic groups of the ligands, (c) 20-60% by weight of crosslinkingmonomers which are capable of free-radical polymerization and have twoor more ethylenically unsaturated polymerizable groups, and with theproviso that a), b) and c) add up to 100% by weight, and wherein thediluent is a mixture of methanol and water in the ratio from 1:1.0 to1:4.0, and wherein the monomer phase is dispersed to droplets in acontinuous phase comprising an organic solvent comprising an aliphatichydrocarbon having 5-7 carbon atoms, and where the ratio of monomerphase to continuous phase is from 1:2.0 to 1:4.0, and in this formundergo free-radical polymerization in the presence of a polymerizationinitiator and of a protective colloid, with the proviso that the ratioof the monomers to the diluent is from 1:1.7 to 1:2.4, and wherein thecarrier polymer materials are coated with penicillin amidase to form thecoated carrier.
 2. The process as claimed in claim 1, wherein themonomers are: a) acrylamide and/or methacrylamide b) glycidylmethacrylate and/or allyl glycidyl ether c) N,N′-methylenebisacrylamideor N,N′-methylene-bismethacrylamide.
 3. The process as claimed in claim1, wherein the organic solvent is cyclohexane.
 4. The process as claimedin claim 1, wherein the penicillin amidase is derived from E. coli.
 5. Aprocess for the synthesis of cephalexin, comprising: contacting thereactants for cephalexin with a carrier material, wherein the carriermaterial comprises crosslinked hydrophilic carrier polymer materialswhich are able to form covalent bonds in a polymer-analogous reactionwith nucleophilic groups of ligands, are in bead form and can beprepared by inverse bead polymerization of a monomer phase whichconsists of monomers and a diluent, wherein the monomers are: (a) 5-40%by weight of hydrophilic monomers which are capable of free-radicalpolymerization, have a vinyl group and form at least 10% strengthaqueous solutions at room temperature, (b) 30-50% by weight of monomerswhich are capable of free-radical polymerization and have a vinyl groupand an additional functional group which is able to enter into covalentbonds in a polymer-analogous reaction with the nucleophilic groups ofthe ligands, (c) 20-60% by weight of crosslinking monomers which arecapable of free-radical polymerization and have two or moreethylenically unsaturated polymerizable groups, and with the provisothat a), b) and c) add up to 100% by weight, and wherein the diluent isa mixture of methanol and water in the ratio from 1:1.0 to 1:4.0, andwherein the monomer phase is dispersed to droplets in a continuous phasecomprising an organic solvent comprising an aliphatic hydrocarbon having5-7 carbon atoms, and where the ratio of monomer phase to continuousphase is from 1:2.0 to 1:4.0, and in this form undergo free-radicalpolymerization in the presence of a polymerization initiator and of aprotective colloid, with the proviso that the ratio of the monomers tothe diluent is from 1:1.7 to 1:2.4.
 6. The process as claimed in claim1, wherein the monomers a) are methacrylamide.
 7. The process as claimedin claim 1, wherein the monomers b) are glycidyl methacrylate and allylglycidyl ether.
 8. The process as claimed in claim 1, wherein themonomers c) are N,N′-methylene-bismethacrylamide.
 9. The process asclaimed in claim 1, wherein the carrier polymer material has a size offrom 50 to 500 μm.
 10. The process as claimed in claim 1, wherein thecarrier polymer material has a size of from 120 to 250 μm.
 11. Theprocess as claimed in claim 5, wherein the monomers a) aremethacrylamide.
 12. The process as claimed in claim 5, wherein themonomers b) are glycidyl methacrylate and allyl glycidyl ether.
 13. Theprocess as claimed in claim 5, wherein the monomers c) areN,N′-methylene-bismethacrylamide.
 14. The process as claimed in claim 5,wherein the carrier polymer material has a size of from 50 to 500 μm.15. The process as claimed in claim 5, wherein the carrier polymermaterial has a size of from 120 to 250 μm.